Artificial Target Cells for in-vitro CAR Cytotoxicity and ADCC validation
Artificial target cells lines with improved distinction between background killing and ADCC and/or CAR-mediated killing are presented. In some embodiments, the artificial cells are recombinant SUP-B15 cells expressing target antigens that are recognized by a CAR, and/or a bispecific engager, or a therapeutic antibody.
1 . A recombinant target cell, comprising:
a recombinant nucleic acid comprising a sequence that encodes a transmembrane antigen wherein the sequence is operably coupled to a promoter to express the sequence that encodes the transmembrane antigen; and
wherein the cell exhibits equal or less than 20% spontaneous lysis in the presence of a CAR-T or an NK cell at an effector cell to target cell ratio of 1 or less.
2 . The recombinant target cell of claim 1 , wherein the transmembrane antigen is a tumor associated antigen, a tumor specific antigen, or a patient- and tumor specific antigen.
3 . The recombinant target cell of claim 1 , wherein the transmembrane antigen is an antigen present on a solid tumor.
4 . The recombinant target cell of claim 1 , wherein the transmembrane antigen is an antigen present on a liquid tumor.
5 . The recombinant target cell of claim 1 , wherein the transmembrane antigen is PD-L1, CD33, CD123, HER-2, or CD20.
6 . The recombinant target cell of claim 1 , wherein the recombinant nucleic acid is a recombinant lentiviral expression vector.
7 . The recombinant target cell of claim 1 , wherein the recombinant nucleic acid is a recombinant RNA.
8 . The recombinant target cell of claim 1 , wherein the cell is a leukemia cell.
9 . The recombinant target cell of claim 8 , wherein the leukemia cell is SUP-B15.
10 . The recombinant target cell of claim 9 , wherein the cell exhibits equal or less than 15% spontaneous lysis in the presence of CAR-T or NK cell at an effector cell to target cell ratio of 10 or less.
11 . The recombinant target cell of claim 9 , wherein the cell exhibits equal or less than 10% spontaneous lysis in the presence of CAR-T or NK cell at an effector cell to target cell ratio of 1 or less.
12 . The recombinant target cell of claim 1 , wherein the effector cell is a CAR-T cell.
13 . The recombinant target cell of claim 1 , wherein the effector cell is an NK cell.
14 . A method of quantifying CAR or ADCC mediated cell killing, comprising:
providing or generating a recombinant target cell as claimed in claim 1 ;
providing or generating a CAR-T or an NK cell;
incubating the recombinant target cell and the CAR-T or NK cell at a predetermined effector cell to target cell ratio; and
quantifying the ADCC or CAR-mediated cell killing.
15 . The method of claim 14 wherein the recombinant target cell exhibits equal or less than 15% spontaneous lysis in the presence of CAR-T or NK cell at an effector cell to target cell ratio of 10 or less or wherein the recombinant target cell exhibits equal or less than 10% spontaneous lysis in the presence of CAR-T or NK cell at an effector cell to target cell ratio of 1 or less.
16 . The recombinant target cell of claim 14 , wherein the transmembrane antigen expressed by the recombinant target cell is a tumor associated antigen, a tumor specific antigen, or a patient- and tumor specific antigen.
17 . The method of claim 16 wherein the transmembrane antigen is PD-L1, CD33, CD123, HER-2, or CD20.
18 . The method of claim 14 wherein the step of incubating is performed while the recombinant target cell and the CAR-T or NK cell are in suspension.
19 . The method of claim 14 wherein the method is used to screen an antibody library against the transmembrane antigen.
20 . The recombinant target cell of claim 14 , wherein the step of quantifying the ADCC or CAR-mediated cell killing is performed using a flow cytometry-based cytotoxicity or ADCC assay.